5-HT stimulation of heart rate in Drosophila does not act through cAMP asrevealed by pharmacogenetics

Zana R. Majeed,1,2 Charles D. Nichols,3 and Robin L. Cooper1

1Department of Biology, University of Kentucky, Lexington, Kentucky; 2Department of Biology, College of Science, Universityof Salahaddin, Erbil, Iraq; and 3Department of Pharmacology and Experimental Therapeutics, Louisiana State UniversityHealth Sciences Center, New Orleans, Louisiana

Submitted 22 July 2013; accepted in final form 25 September 2013

Majeed ZR, Nichols CD, Cooper RL. 5-HT stimulation of heartrate in Drosophila does not act through cAMP as revealed bypharmacogenetics. J Appl Physiol 115: 1656–1665, 2013. First pub-lished October 3, 2013; doi:10.1152/japplphysiol.00849.2013.—Thefruit fly, Drosophila melanogaster, is a good experimental organismto study the underlying mechanism of heart rate (HR) regulation. It isalready known that many neuromodulators (serotonin, dopamine,octopamine, acetylcholine) change the HR in Drosophila melano-gaster larvae. In this study, we investigated the role of cAMP-PKAsignaling pathway in HR regulation and 5-HT positive chronotropicaction. In order to obtain insight into the 5-HT mechanism of actionin larvae cardiomyocytes, genetic and pharmacological approaches wereused. We used transgenic flies that expressed the hM4Di receptor [de-signer receptors exclusively activated by designer drugs (DREADDs)] asone tool. Our previous results showed that activation of hM4Direceptors (modified muscarinic acetylcholine receptors) decreases orarrests the heart from beating. In this study, it was hypothesized thatthe positive chronotropic effect of serotonin [5-hydroxytryptamine(5-HT)] are mediated by serotonin receptors coupled to the adenylylcyclase pathway and downstream cAMP and PKA activity. Activationof hM4Di by clozapine-N-oxide (CNO) was predicted to block theeffects of serotonin by inhibiting adenylyl cyclase activity throughG�i pathway activation. Interestingly, we found here that manipula-tion of adenylyl cyclase activity and cAMP levels had no significanteffect on HR. The ability of hM4Di receptor activation to slow or stopthe heart is therefore likely mediated by activation of GIRK channelsto produce hyperpolarization of cardiomyocytes, and not throughinhibition of adenylyl cyclase.

5-HT; M4D receptor; CNO; adenylyl cyclase; PKA

THE HEART IN THE FRUIT FLY, DROSOPHILA MELANOGASTER, IS A MODEL

for investigating the underlying mechanism of cardiac diseaseswhich are related to human cardiac disorders, such as congen-ital heart diseases (39) and dilated cardiomyopathy (25). Al-though, the Drosophila heart is simple and consists of a hearttube at the caudal region and anterior aortic region (9), it hasbeen established as a model system to study the ionic basis ofmyocardiocytes (15). The myogenic heart of Drosophila larvaealso is useful to examine modulators and their mode of actions,because modulators directly act on the myocardiocytes as thereis no direct innervation on the pacemaker region. It has beenpreviously shown that Ca2� and K� ions play a crucial role ingeneration of the cardiac action potential. Inhibition of L-typecalcium channels or potassium channels by specific blockerssignificantly reduce the heart rate (HR) whereas the Na�

channel blocker, tetrodotoxin, does not have any effect on HR

in Drosophila (18). To investigate the underlying mechanismsof modulation, ionic and second messenger cascades need to bedetermined.

Some neuromodulators and neurotransmitters, like sero-tonin, dopamine, octopamine, and acetylcholine are known tomodulate heart performance in Drosophila melanogaster lar-vae (11, 20, 37). 5-Hydroxytryptamine (5-HT) is an essentialneuromodulator that has many behavioral and physiologicalfunctions in the fly, such as learning and memory (22), court-ship and mating (3), cardiac rate modulation (11, 41), andmodulation of sensory-motor circuits (13). Moreover, 5-HT isassumed to be a modulator with an old evolutionary historybecause it is found in simple as well as in complex animals andeven plants (2). There are four characterized 5-HT receptorgenes in the Drosophila melanogaster genome, 5-HT1ADro,5-HT1BDro, 5-HT2Dro, and 5-HT7Dro (3, 22, 24, 30). Re-cently, another 5-HT2Dro receptor subtype, 5-HT2BDro, hasbeen identified in Drosophila (17). 5-HT1ADro, 5-HT1BDroinhibits adenylyl cyclase (AC) activity (36); 5-HT7Dro in-creases AC activity (40). Exogenous 5-HT application insemi-intact larvae increases the HR, but the underlying signal-ing mechanism has yet to be elucidated. However, we haveshown that activation of 5-HT2Dro receptor mediates thepositive chronotropic effect of 5-HT in Drosophila larval heart(Majeed ZR, Stacy A, Cooper RL, unpublished observations).

It is obvious that the Drosophila and vertebrates are differentmorphologically; notwithstanding, Drosophila and vertebratesuse similar molecular pathways underlying cardiac develop-ment (7). Further, Drosophila and vertebrate hearts sharecrucial physiological and dynamic aspects, for example, car-diac output, and rate and duration of systole or diastole (10).The rationale for Drosophila larval heart study is to decode theundiscovered aspects of cardiac physiology and pathophysiol-ogy which in turn can be extended to the physiology of heartsin other animals, including humans.

In this study, we sought to elucidate the signaling pathwayunderlying the positive chronotropic effects of 5-HT in Dro-sophila melanogaster larvae by utilizing classical pharmacol-ogy with incorporation of a pharmacogenetic approach usingdesigner receptors exclusively activated by designer drugs(DREADD) receptors (4). DREADDs are powerful new toolsthat allow a high degree of spatial and temporal control ofneuronal and effector pathway activity. Significantly, DREADDcontrol is reversible, and requires no specialized equipment.We used the UAS-Gal4 binary expression system (8) to ex-press hM4Di receptors, which are positively coupled to G�i inmuscle fibers. The hM4Di is a modified human muscarinicacetylcholine M4 receptor, mutated such that it no longer hasaffinity for the native ligand, acetylcholine. Instead, this engi-

Address for reprint requests and other correspondence: R. L. Cooper, Dept.of Biology, 675 Rose St., Univ. of Kentucky, Lexington, KY 40506-0225(e-mail: RLCOOP1@pop.uky.edu).

J Appl Physiol 115: 1656–1665, 2013.First published October 3, 2013; doi:10.1152/japplphysiol.00849.2013.

8750-7587/13 Copyright © 2013 the American Physiological Society http://www.jappl.org1656

neered receptor has high affinity for a chemical that is consid-ered physiologically inert, clozapine-N-oxide (CNO) that hasfull agonist efficacy at DREADD receptors (1, 4, 29). With thisapproach one can rule out off-target effects of the naturalligand to specifically and remotely control effector pathwayactivity in defined target tissues that the DREADD receptor isexpressed in.

In the pupal stage of Drosophila forskolin does not producea change in the HR (21), indicating that cAMP levels are notimportant in the pupa; however, in the pupal stage significantalterations in endocrine function accompanied by significantmorphological changes are occurring, and mechanisms of car-diac function may be different than in the larva. We previouslydemonstrated that 5-HT increases HR in larvae; therefore, weexamine here the potential mechanisms of action of 5-HT inthe larval stage heart. Our working model was that 5-HTactivates G�s, which activates adenylyl cyclase that in turnincreases the level of cAMP. Increased cAMP leads to anincrease in active PKA signaling and positive chronotropicmodulation of the heart. In this scenario, blockade of adenylylcyclase activity with pharmacological methods, or activation ofG�i through activation of hM4Di receptors expressed in theheart, would be predicted to decrease cAMP levels and PKAactivation and block the positive chronotropic effects of 5-HTon the heart. Our data, however, indicate that cAMP levels donot contribute to either modulation of HR or the positivechronotropic effects of 5-HT in the Drosophila heart. It hasbeen reported that forskolin does not noticeably change the HRin P1 pupal stage of Drosophila (21). This confirms thatDrosophila HR in larvae also might not be markedly changedby activation of AC-PKA pathway; although, AC-PKA path-way might increase the contractility of heart.

MATERIALS AND METHODS

Transgenic fly strains. In this study, two fly strains were used,UAS-hM4Di and 24B-GFP (a strain heterozygous for the 24B-GAL4driver element that expresses in all larval muscle, and is also homozy-gous for the UAS-GFP element; from Dr. Charles Nichols, LouisianaState University Health Sciences Center). The UAS-hM4Di strain wascrossed with 24B-GFP strain in order to express hM4Di (G�i)coupled receptor in muscle fibers, including cardiomyocytes. In the F1generation, half the flies contained the 24B-GAL4 expression ele-ment, detected by GFP expression, and the other half did not. TheGFP-positive, hM4Di-expressing larvae were used as the experimen-tal animals, and the non-GFP-positive, non-hM4Di-expressing (butUAS-hM4Di containing) ‘littermates’ were used as controls (back-ground). The flies were reared at room temperature (22°C) in vialscontaining cornmeal-agar-dextrose-yeast medium.

Heart rate measurement. Third instar larvae were dissected in theventral side up position to expose the dorsal vessel (heart) in HL3(saline solution) (NaCl 70 mM, KCl 5 mM, MgCl2.6H2O 20 mM,NaHCO3 10 mM, Trehalose 5 mM, sucrose 115 mM, BES 5 mM, andCaCl2.2H2O 1 mM with the pH adjusted to 7.1). Because heartperformance is very sensitive to pH change, the pH was tightlyregulated and adjusted as needed. Recently, the effects of variousbuffers have been probed to optimize the saline for monitoring heartrate (14). Drugs were applied at various concentrations as indicated inthe Results. The preparation was left for 1 min in saline afterdissection, and then heart beats were counted for the followingminute. Exceptions are indicated on graphs. The difference in the HRbefore and after application of drugs was used to measure the effectsof the various compounds.

Chemicals. All the chemicals, serotonin hydrochloride [5-hydroxy-tryptamine (5-HT)], forskolin, SQ 22,536, N6, 2=-O-Dibutyryl aden-osine 3=,5= cyclic monophosphate sodium salt (dbcAMP), 2=,5= dide-oxyadenosine were purchased from Sigma-Aldrich, St. Louis, MO.Clozapine-N-oxide was kindly synthesized by Dr. David Nichols(Purdue University). 5-HT stock solution (1 mM) was prepared withHL3 saline and immediately before use, a 1 �M serotonin solutionwas made from the stock solution. Forskolin was dissolved in 1%dimethyl sulfoxide (DMSO) to obtain a 1 mM stock solution. Thefinal concentration of DMSO was less than 0.1% (roughly about0.03%) in 30 �M forskolin and 0.5% in 100 �M forskolin. 300 �Mand 1 mM of dbcAMP were also prepared with HL3 saline. 200 �Mof SQ 22,536 was made with saline from a 1 mM stock solution. A1-mM dideoxyadenosine stock solution was made in DMSO, then 50�M (0.04% final DMSO concentration) and 500 �M (0.2% finalDMSO concentration) with dilutions in saline.

Statistical analysis. All data are expressed as mean � SEM. Thepaired t-test (before and after) was used to compare the difference ofHR change after exchanging solution with saline containing chemi-cals. T-test was used to compare between background (UAS-hM4Di)and hM4Di expressing (UAS-hM4DiX24B-Gal4) larval HRs (Sigma-Plot version 11.0). P � 0.05 is considered as statistically significant.The number of asterisks are considered as P � 0.05 (*), P � 0.02(**), and P � 0.001 (***).

RESULTS

Effects of media change on heart rate. We previously foundthat the larval HR is sensitive to the physical exchange of thebathing media (9). In order to control for the exchange of thebathing media when exposing the heart to various compounds,a series of control experiments of saline bath exchanges wasconducted. Also, because some of the drugs contained smallconcentrations of DMSO, the effect of low concentrations ofDMSO alone and the effect of switching the DMSO bathingmedia were examined (Fig. 1). In addition, the HR was mea-sured with an additional exchange of the bathing media twicefor the same period as the first one, left for 1 min, and thenexamined for the following minute. Although the larvae werealready exposed to saline during the dissection, there was stillan average of 20 beats per minute (BPM) increase in HR withthe first saline change. After another 2 min the heart was not assensitive to the exchange of the saline, as only an average of 10BPM was noted (Fig. 1B). Exchanging bathing media thatcontained DMSO did not produce as large of an increase in HRas saline. The second exchanges also resulted in a smalleralteration in HR (Fig. 1D).

CNO concentration dependently inhibits heart rate in hM4Diexpressing larvae. Increasing concentrations of CNO (10 nM,100 nM, and 1 �M) were applied to the exposed heart ofsemi-intact larval preparations. Application of CNO decreasesthe HR in a concentration dependent manner in the UAS-M4DX 24B-GFP larvae (Fig. 2). CNO application at the highestconcentration does not significantly change the HR in thenonexpressing UAS-M4D (control) larvae (Fig. 2). In thisstudy, 500 nM CNO was chosen to use for the primaryexperiments because 1 �M stopped the heart from beating inall UAS-M4D expressing preparations. We observed that mostof the hearts started to beat again when the saline � CNO wasexchanged with saline only (not shown).

The presence of CNO to activate hM4Di in expressinglarvae blocks the positive chronotropic effect of 5-HT. In fliesthat do not express hM4Di receptors (background), 5-HT

1657Pharmacogenetic Inhibition of the Larval Heart • Majeed ZR et al.

J Appl Physiol • doi:10.1152/japplphysiol.00849.2013 • www.jappl.org

markedly increases the HR, even in the presence of CNO(Fig. 3, A and C). These results confirm the positive chrono-tropic effects of 5-HT and that CNO does not block the plasmamembrane receptor binding site for 5-HT. Only when hM4DiDREADD receptor expression was induced did CNO have aneffect on the positive chronotropic effect of 5-HT (Fig. 3, B andC). We observed that CNO by itself slightly decreased HR inbackground larvae (Fig. 3, A and C). This may be due to leakyexpression of the UAS-GAL4 element present in the back-ground genotype rather than a direct effect of CNO on theheart. When the CNO � 5-HT containing saline was ex-changed with saline (4¡5), the HR significantly increased inboth background and hM4Di-expressed larvae. This might bedue to the 5-HT residual effect on the HR. The increase in HRon this final wash was 2-fold greater in the hM4Di expressing

larvae, suggesting a rebound effect occurring from the removalof the repressing CNO mediated hM4Di signaling.

After 5-HT is applied to increase HR, the addition of CNOreverses the positive chronotropic effects of 5-HT. 5-HT wasapplied prior to exposure of CNO in order to see if CNO canalso reverse the effects of 5-HT in addition to block, asdemonstrated above. 5-HT markedly increases the HR (Fig. 4).The addition of CNO reversed the effects of 5-HT (Fig. 4).When CNO � 5-HT saline was exchanged with saline alone(4¡5), the HR increased again, as observed in the experimentsshown in Fig. 3, indicating a possible rebound effect fromremoval of the repressing hM4Di signaling.

CNO blocks the effects of forskolin. In order to know moreabout the effect of hM4Di expression on cardiac muscle fibersecond messengers, forskolin, an AC activator was examined.In the presence of CNO forskolin increased the HR in back-ground larvae (Fig. 5A). The presence of CNO blocked thepositive chronotropic effect of forskolin in hM4Di-expressinglarvae. Furthermore, not only was the HR completely arrestedby CNO application, but forskolin was unable to overcome thisblockade (Fig. 5, B and C).

Forskolin (30 �M) alone was observed to increase the HR(Fig. 6). The subsequent addition of CNO had no inhibitingeffect in the background larva but dramatically halted the HRin the hM4Di expressing larvae (Fig. 6). Since forskolin wasnot able to overcome the effects of hM4Di activation this mayindicate that cAMP levels alone are not the primary mediatorof HR. The effect of forskolin did not show a significantdifference between two genotypes (Fig. 6C).

A higher concentration of forskolin (100 �M) was used todetermine whether or not the 30 �M concentration was max-imal or near maximal and thus producing a saturating effect.We also incubated with the higher concentration of forskolinfor 10 min to observe if there was a time-dependent effectpresent on heart rate for forskolin exposure compared with anacute exposure. We observed that forskolin at 100 �M in-creased the HR at the beginning; however, it was not greater

Fig. 1. A: The effect of changing saline with salineon larval heart rate in UAS-M4D X 24B-GFP flies,n � 9. B: Difference in heart rate before and afterchanging solution. C and D: The effect of changingsaline containing DMSO with saline containingDMSO on larval heart rate in UAS-M4D X 24B-GFP flies; n � 10. P � 0.05 (*), P � 0.02 (**).

Fig. 2. Concentration response curve for clozapine-N-oxide (CNO) at hM4Direceptors on the larval heart rate. The absolute change in rate [beats per minute(BPM)] is shown P � 0.001 (***).

1658 Pharmacogenetic Inhibition of the Larval Heart • Majeed ZR et al.

J Appl Physiol • doi:10.1152/japplphysiol.00849.2013 • www.jappl.org

than that of 30 �M, and no further increase the HR occurredduring the 10 min incubation time (Fig. 7, A and B), indicatingthat our original 30 �M concentration was likely saturating forforskolin.

To further probe the role of cAMP, we used the AC inhibitorSQ 22,536 (SQ). Application of SQ had no effect in the firstminute (Fig. 8). Because no inhibitory effect of SQ on the HRwas observed within the first few minutes, we prolonged theinterval to 9 min. At the ninth minute of incubation the HRdecreased significantly (P � 0.02), but it did not stop (Fig. 8).This decrement in HR might be due to the lengthy incubationperiod, rather than the drug. However, it was shown if thesemi-intact heart preparation (Canton-S flies) that were incu-

bated inside the saline (pH 7.00) for 10 min the HR do notshow a marked change (data not shown). Taken together, theseresults show that SQ has a slightly inhibitory effect on the HR.When the SQ solution was exchanged with SQ � 5-HTsolution, HR significantly increased (Fig. 8). These resultsindicate that SQ cannot override the positive chronotropiceffect of 5-HT. Either the SQ is not effective in blockingDrosophila AC as it is for mammalian AC, or perhaps manip-ulating cAMP levels alone is not sufficient to mediate anincrease in HR, in agreement with our results using forskolin.

We next tried another AC inhibitor, dideoxyadenosine. Ap-plication of dideoxyadenosine (50 and 500 �M) did not pro-duce a noticeable change in HR in hM4Di expressing larvae

Fig. 3. Effect of activation of hM4Di recep-tors on the action of 5-HT in larval heart.A: Background UAS-M4D, n � 10. B: UAS-M4D X 24B-GFP, n � 10. C: Comparisonbetween background (dark grey) and M4Dexpressing group (light grey). The differ-ences between genotypes are represented byNS (not significant) or asterisk(s) above thelines. The differences, which are due to thechanging of one solution with another one inthe same genotypes, are represented by NSor asterisk(s) above the bars. P � 0.05 (*),P � 0.02 (**), P � 0.001 (***).

Fig. 4. A: Effect of 5-HT application plushM4Di activation on larval heart rate (UAS-M4D X 24B-GFP); n � 9. B: The difference inheart rate before and after change of solution.P � 0.02 (**), P � 0.001 (***).

1659Pharmacogenetic Inhibition of the Larval Heart • Majeed ZR et al.

J Appl Physiol • doi:10.1152/japplphysiol.00849.2013 • www.jappl.org

(Fig. 9 and 10). By the second minute HR showed a very slightdecrease in hM4Di expressing larvae. However, when dide-oxyadenosine (50 or 500 �M) solution was exchanged withdideoxyadenosine � 5-HT, a marked increase in the HRoccurred (Fig. 9 and 10).

In a different approach, we attempted to manipulate cAMPlevels by using the cAMP analog dbcAMP, which both inhibitsphosphodiesterase and mimics the effects of cAMP on down-stream effectors like PKA. dbcAMP is resistant to cleaving by

phosphodiesterase (19). Application of dbcAMP (300 �M)does not change the HR in background larvae (Fig. 11, A andC). Because no effect was observed with the initial exposure ofdbcAMP on the HR, the HR was measured for the followingsecond minute. Again, there was no significant change in HRwith a longer exposure to dbcAMP (Fig. 11, A and C). Whenthe dbcAMP solution was exchanged with dbcAMP � CNOsolution, HR slightly increased in background larvae (Fig. 11,A and C). Application of dbcAMP slightly decreased HR in

Fig. 5. Effect of hM4Di activation plus fors-kolin application on larval heart rate. A: Back-ground strain UAS-M4D and heart rate withsolution changing; n � 6. B: Change of heartrate in UAS-M4D X 24B-GFP, n � 5, with thesame solutions as in A. C: Comparison be-tween background (dark grey) and M4D ex-pressing group on the effect of CNO and for-skolin (light grey). The differences betweengenotypes are represented by NS (not signifi-cant) or asterisk(s) above the lines. The differ-ences, which are due to the changing onesolution with another one in the same geno-types, are represented by NS or asterisk(s)above the bars. P � 0.05 (*), P � 0.02 (**),P � 0.001 (***).

Fig. 6. Effect of forskolin plus hM4Di acti-vation on larval heart rate. A: Background,UAS-M4D, n � 7. B: UAS-M4D X 24B-GFP, n � 5. C: Comparison between back-ground (dark grey) and M4D expressinggroup (light grey). The differences betweengenotypes are represented by the NS (notsignificant) or asterisk(s) above the lines.The differences, which are due to the chang-ing one solution with another one in thesame genotypes, are represented by NS orasterisk(s) above the bars. P � 0.02 (**),P � 0.001 (***).

1660 Pharmacogenetic Inhibition of the Larval Heart • Majeed ZR et al.

J Appl Physiol • doi:10.1152/japplphysiol.00849.2013 • www.jappl.org

hM4Di expressing larvae (Fig. 11, B and C). When the db-cAMP solution was exchanged with the dbcAMP � CNOsolution, the HR dramatically decreased in the hM4Di express-ing larvae (Fig. 11, B and C). dbcAMP did not have asignificant difference between two genotypes.

Because there was no significant change after application of300 �M dbcAMP, a higher concentration of dbcAMP (1 mM)was used. Application of dbcAMP at this higher concentrationonly slightly decreased the HR in the first and second minute(Fig. 12) to the same degree as with the lower concentration(Fig. 11). However, when the dbcAMP solution was ex-changed with dbcAMP � CNO, the HR markedly decreased(Fig. 12). Changing dbcAMP � CNO solution with salinealone showed a marked increase in HR (Fig. 12).

DISCUSSION

Drosophila has been established to be a good model for thestudy of cardiac physiology in part because of malleablegenetics and conserved development with mammalian heart(33). Moreover, fly and mammalian hearts use many of thesimilar signaling pathways during development (39). In mam-mals, 5-HT receptor dysfunction or overactivation leads toabnormal cardiac development and physiological disturbance(23, 28). Clearly, 5-HT and proper activation of relevant 5-HTreceptors is critical for normal cardiac development and func-tion in mammals (5). 5-HT receptors are all G-protein coupled

receptors (GPCRs), with the exception of the 5-HT3 receptorthat is a ligand gated ion channel (31). Modified DREADDGPCR receptors have been shown to be excellent tools tofurther understand the function of specific GPCRs, like sero-tonin receptors, and their signaling pathways in normal phys-iological processes in both fly and mammals (4, 29). Forexample, the hM4D1 DRAEDD receptor we have used herehas also been used to understand the role of serotonin inrespiratory control (34) and to determine key structural com-ponents of the native muscarinic M4 acetylcholine receptoritself (27). These pharamcogenetic approaches have opened upnew avenues to further understand the roles of GPCR receptorsin physiology and are promising tools to obtain additionalinsight into underlying molecular mechanisms of human dis-eases.

Here, we initiated studies to elucidate effector pathwaysunderlying HR regulation in the Drosophila heart and tofurther explore the positive chronotropic role of serotonin onthe Drosophila larva heart. Our original hypothesis was thatDrosophila larva HR, like mammalian HR, was governed byadenylyl cyclase activity and its downstream effectors andsecond messengers. Further, that the positive chronotropiceffect of 5-HT was due to positive modulation of adenylylcyclase activity. To address this hypothesis, we used bothpharmacological and pharmocogenetic approaches using acombination of drugs and the modified mammalian G-protein

Fig. 7. Effect of 100 �M forskolin on larvalheart rate. A: Background, UAS-M4D, n �8, heart rate of individual preparations.B: Background, UAS-M4D, n � 8, showsthe change in heart rate after application offorskolin. P � 0.02 (**), P � 0.001 (***).

Fig. 8. A and B: Effect of SQ 22,536 andSQ � 5-HT combination on larval heart rate(UAS-M4D X 24B-GFP); n � 10. P � 0.05(*), P � 0.02 (**), P � 0.001 (***).

1661Pharmacogenetic Inhibition of the Larval Heart • Majeed ZR et al.

J Appl Physiol • doi:10.1152/japplphysiol.00849.2013 • www.jappl.org

coupled receptor (hM4Di, coupled to G�i) to provide aninsight into potential signaling pathways underlying HR andthe effects of 5-HT in Drosophila larval heart. We initiallyobserved that activation of hM4Di can slow or stop the heart.Moreover, that hM4Di activation can override the positivechronotropic effect of 5-HT. These observations were consis-tent with our hypothesis, with the prediction that hM4Diactivation would activate G�i, reduce adenylyl cyclase activ-ity, and slow the heart.

It has been shown, however, that forskolin does not notice-ably change the HR in P1 pupal stage of Drosophila (21). Weextended the investigation in larvae and showed, in general,manipulation of adenylyl cyclase activity and cAMP levelswith the dbcAMP, SQ 22,536 and dideoxyadenosine did nothave a noticeable impact on the HR. In rodents adenosineanalogs can retard the positive chronotropic effect of norepi-nephrine (35). There was a positive effect of forskolin; how-ever, the effect was only slight. Also, dbcAMP slightly de-creased the HR. This may be due to activation of targets bycAMP that could bring about ion channel activation, such asK� channels. This might result in depolarization of the pace-maker cell membrane potential. Further, adding dbcAMP is notthe same as stimulating AC as there are various isoforms of ACthat might be differentially activated by forskolin whereasaddition of dbcAMP maintains a high level of cAMP, bypass-ing activation of AC.

Our findings indicate that cAMP signaling does not contributeto regulation of HR in Drosophila larvae as it does in mammals,and that cAMP is not a component of the positive chronotropiceffects of 5-HT in the fly heart. Supporting this conclusion is that5-HT is still able to produce a large increase in HR in the presenceof AC inhibitors. As an additional tool to probe mechanismsunderlying HR, we employed the designer receptor hM4Di, whichis positively coupled to G�i signaling in both mammals and thefly, as well as the G-coupled inwardly rectifying potassium chan-nel (GIRK) to produce silencing. For example, CNO activation ofhM4Di in mammalian systems results in the hyperpolarizationand electrical silencing of hippocampal neurons in culture (1). Wepreviously observed that CNO significantly slowed or stopped theheart when applied to Drosophila hearts expressing hM4Di re-ceptors (4) and observe the same effect here. Significantly, phar-macological manipulation of AC or cAMP levels in the presenceof activated hM4Di had no effect on the negative chronotropiceffects of activated hM4Di. Forskolin was unable to override therepressing effects of CNO. Together, the responses likely indicatethat the main effects of hM4Di activation on HR are probablymediated through G�� activation of GIRK channels with subse-quent hyperpolarization and silencing of cardiomyocytes ratherthan through negative modulation of cAMP levels through G�isignaling.

When we applied 5-HT to the larvae preparations, weobserved that 5-HT markedly increased the HR, in agreement

Fig. 9. A: Effect of dideoxyadenosine anddideoxyadenosine � 5-HT combination onlarval heart rate (UAS-M4D X 24B-GFP),n � 8. B: The difference in heart rate beforeand after change of solution. P � 0.02 (**),P � 0.001 (***).

Fig. 10. A: Effect of a high concentration ofdideoxyadenosine and dideoxyadenosine �5-HT combination on larval heart rate (UAS-M4D X 24B-GFP), n � 10. B: The differencein heart rate before and after change of solu-tion. P � 0.05 (*), P � 0.001 (***).

1662 Pharmacogenetic Inhibition of the Larval Heart • Majeed ZR et al.

J Appl Physiol • doi:10.1152/japplphysiol.00849.2013 • www.jappl.org

with our earlier studies. Our original hypothesis was that 5-HTactivates AC, which in turn lead to the activation of PKA andphosphorylation of Ca2� channels that lead to inward Ca2�

currents and contraction. If this were correct, then the positivechronotropic effects of 5-HT action could be blocked bydecreasing AC activity or levels of cAMP. Since our multipleattempts at manipulation of AC and cAMP levels had no orlittle effect on HR indicate that HR in the fly is in fact notsubstantially mediated by cAMP. The inhibitory effect ofactivation of hM4Di we observed is therefore likely mediatedthrough G�� activation of GIRK channels and silencing ofcardiomyocytes, rather than inhibition of AC through G�i todecrease cAMP levels and PKA activity.

Toward understanding the mechanism of action of serotonin onthe heart, it needs to be determined if there may be different 5-HT

receptors on heart muscle fibers acting through multiple signalingmechanisms. The known fly 5-HT receptors are all G-proteincoupled receptors: 5-HT1ADro and 1BDro inhibit adenylyl cy-clase through activation of G�i; 5-HT7Dro activates adenylylcyclase through G�s activation; 5-HT2Dro signaling pathway hasnot been identified yet (6, 32, 36). A remaining possibility for thepositive chronotropic effect of 5-HT could be that 5-HT2Droreceptors are mediating HR. 5-HT2 receptors couple with G�qand activation of phospholipase C (PLC). PLC cleaves PIP2 toIP3 and diacylglycerol (DAG). IP3 leads to increases in intracel-lular Ca2� (16), which could lead to contraction. However, wepreviously demonstrated that mutation or misexpression of the5-HT2Dro receptor does not have a dramatic effect on Drosophilalarval HR (12). A second 5-HT2Dro receptor has recently beenreported (17), and it may be this receptor mediating the effects if

Fig. 11. Effect of dbcAMP and dbcAMp�CNOcombination on larval heart rate. A: Back-ground UAS-M4D, n � 11. B: UAS-M4D X24B-GFP, n � 15. C: Comparison betweenbackground (dark grey) and M4D expressinggroup (light grey). The differences betweengenotypes are represented by NS (not signifi-cant) or asterisk(s) above the line. The differ-ences, which are due to changing one solutionwith another one in the same genotypes, arerepresented by NS or asterisk(s) above thebars. P � 0.05 (*), P � 0.02 (**), P � 0.001(***).

Fig. 12. Effect of dbcAMP (1 mM) and db-cAMP � CNO combination on larval heartrate. UAS-M4D X 24B-GFP (n � 10). B: Thedifference in heart rate before and after changeof solution.P � 0.05 (*), P � 0.02 (**), P �0.001 (***).

1663Pharmacogenetic Inhibition of the Larval Heart • Majeed ZR et al.

J Appl Physiol • doi:10.1152/japplphysiol.00849.2013 • www.jappl.org

G�q pathways are involved. Recently, a pharmacological studyhas shown that activation of 5-HT2 receptor by 5-HT2 agonistincreases HR and ketanserin, which is 5-HT2 antagonist, mark-edly decreases the action of 5-HT on HR in Drosophila larvae(Majeed ZR, Stacy A, Cooper RL, unpublished observations).Therefore, we speculate that a PLC pathway might mediate thepositive chronotropic effect of 5-HT. However, to confirm thissuspicion, various pharmacological agents should be used tomanipulate the PLC pathway to observe how it effects 5-HTaction related to HR. Another possibility is that 5-HT1ADro,5-HT1BDro, and 5-HT7Dro receptors are mediating HR throughcoupling to other effector pathways than adenylyl cyclase. GPCRscouple to a wide range of effectors, and certain drugs and naturalligands can activate these pathways differentially through func-tional selectivity (38). In this scenario the 5-HT1ADro or5-HT7Dro receptors could mediate HR through noncanonicalpathways yet to be determined. In summary, we present evidencehere that the positive chronotropic effect of 5-HT in the Drosoph-ila larva HR is not governed by the adenylyl cyclase signalingpathway, and that the negative chronotropic effects of hM4Diactivation are likely due to electrical silencing rather than negativemodulation of AC.

GRANTS

This work was funded by National Institute of Mental Health GrantR01MH083689 (C.D.N), Higher Committee for Education Development(HCED) in Iraq (Z.R.M), and personal funds (R.L.C).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: Z.R.M. and R.L.C. performed experiments; Z.R.M.analyzed data; Z.R.M., C.D.N., and R.L.C. interpreted results of experiments;Z.R.M. prepared figures; Z.R.M., C.D.N., and R.L.C. drafted manuscript;Z.R.M., C.D.N., and R.L.C. edited and revised manuscript; Z.R.M., C.D.N.,and R.L.C. approved final version of manuscript; C.D.N. and R.L.C. concep-tion and design of research.

REFERENCES

1. Armburster BN, Li X, Pausch MH, Herlitze S, Roth BL. Evolving thelock to fit the key to create G protein-coupled receptors potently activatedby an inert ligand. Proc Natl Acad Sci USA 104: 5163–5168, 2007.doi:10.1073/pnas.0700293104.

2. Azmitia EC. Serotonin and brain: Evolution, neuroplasticity, and homeo-stasis. Int Rev Neurobiol 77: 31–56, 2007.

3. Becnel J, Johnson O, Luo J, Nässel DR, Nichols CD. The serotonin5-HT7Dro receptor is expressed in the brain of Drosophila, and is essentialfor normal courtship and mating. PLoS One 6(6): e20800, 2011. doi:10.1371/journal.pone.0020800.

4. Becnel J, Johnson O, Majeed ZR, Tran V, Yu B, Roth BL, Cooper RL,Nichols CD. A pharmocogenetic approach for controlling behavior, neuronalsignaling, and physiology in Drosophila. Cell Reports 4(5): 1049–1059, 2013.doi:10.1016/j.celrep.2013.08.003.

5. Berger M, Gray JA, Roth BL. The expanded biology of serotonin. Annu RevMed 60: 355–366, 2009. doi:10.1146/annurev.med.60.042307.110802.

6. Blenau W, Baumann A. Molecular and pharmacological properties ofinsect biogenic amine receptors: Lessons from Drosophila melanogasterand Apis mellifera. Arch Insect Biochem Physiol 48(1): 13–38, 2001.

7. Bodmer R, Venkatesh TV. Heart development in Drosophila and verte-brates: Conservation of molecular mechanisms. Dev Genet 22(3): 181–186, 1998.

8. Brand AH, Perrimon N. Targeted gene expression as a means of alter-ing cell fates and generating dominant phenotypes. Development 118(2):401–415, 1993.

9. Cammarato A, Ahrens CH, Alayari NN, Qeli E, Rucker J, Reedy MC,Zmasek CM, Gucek M, Cole RN, Van Eyk JE, Bodmer R, O’Rourke

B, Bernstein SI, Foster DB. A mighty small heart: The cardiac proteomeof adult Drosophila melanogaster. PLoS One 25, 6(4): e18497, 2011.doi:10.1371/journal.pone.0018497.

10. Choma MA, Suter MJ, Vakoc BJ, Bouma BE, Tearney GJ. Physio-logical homology between Drosophila melanogaster and vertebrate car-diovascular systems. Dis Model Mech 4(3): 411–420, 2011. doi:10.1242/dmm.005231.

11. Dasari S, Cooper RL. Direct influence of serotonin on the larval heart ofDrosophila melanogaster. J Comp Physiol B 176(4): 349–57, 2006.doi:10.1007/s00360-005-0058-3.

12. Dasari S, Wang L, Harrison DA, Cooper RL. Reduced and misexpres-sion of 5-HT2 receptors alters development, behavior and CNS activity inDrosophila melanogaster. Int J Zool Res 5(3): 101–114, 2009. doi:10.3923/ijzr.2009.101.114.

13. Dasari S, Cooper RL. Modulation of sensory-CNS-motor circuits by sero-tonin, octopamine, and dopamine in semi-intact Drosophila larva. NeurosciRes 48(2): 221–227, 2004. doi:org/10.1016/j.neures.2003.10.005.

14. deCastro C, Titlow J, Majeed ZR, Cooper RL. Analysis of variousphysiological salines for heart rate, CNS function, and synaptic transmis-sion at neuromuscular junctions in Drosophila melanogaster larvae. JComp Physiol, in press.

15. Desai-Shah M, Papoy AR, Ward M, Cooper RL. Roles of the Sarco-plasmic/Endoplasmic reticulum Ca2�-ATPase, plasma membrane Ca2�-ATPase and Na�/Ca2� exchanger in regulation of heart rate in larvalDrosophila. Open Physiol J 3: 16–36, 2010.

16. Fukami K, Inanobe S, Kanemaru K, Nakamura Y. Phospholipase C isa key enzyme regulating intracellular calcium and modulating the phos-phoinositide balance. Prog Lipid Res 49(4): 429–437, 2010. doi:10.1016/j.plipres.2010.06.001.

17. Gasque G, Conway S, Huang J, Rao Y, Vosshall LB. Small moleculedrug screening in Drosophila identifies the 5HT2A receptor as a feedingmodulation target. Sci Rep 3: srep02120, 2013. doi:10.1038/srep02120.

18. Gu GG, Singh S. Pharmacological analysis of heartbeat in Drosophila. JNeurobiol 28(3): 269–280, 1995.

19. Henion WF, Sutherland EW, Posternak T. Effects of derivatives ofadenosine 3=,5=-phosphate on liver slices and intact animals. BiochimBiophys Acta 148(1): 106–113, 1967.

20. Johnson E, Ringo J, Dowse H. Modulation of Drosophila heartbeat byneurotransmitters. J Comp Physiol B 167: 89–97, 1997.

21. Johnson E, Sherry T, Ringo J, Dowse H. Modulation of the cardiacpacemaker of Drosophila: cellular mechanisms. J Comp Physiol B 172(3):227–36, 2002. doi:10.1007/s00360-001-0246-8.

22. Johnson O, Becnel J, Nichols CD. Serotonin 5-HT1A-like, 5-HT2, and5-HT7 receptors modulate learning and memory in Drosophila. FASEB J)586.11, 2009.

23. Lairez O, Cognet T, Schaak S, Calise D, Guilbeau-Frugier C, Parini A,Mialet-Perez J. Role of serotonin 5-HT2A receptors in the development ofcardiac hypertrophy in response to aortic constriction in mice. J NeuralTransm 120(6): 927–935, 2013. doi:10.1007/s00702-013-1011-3.

24. Luo J, Becnel J, Nichols CD, Nässel DR. Insulin-producing cells in the brain ofadult Drosophila are regulated by the serotonin 5-HT1A receptor. Cell Mol LifeSci 69(3): 471–484, 2012. doi:10.1007/s00018-011-0789-0.

25. Ma L, Bradu A, Podoleanu AG, Bloor JW. Arrhythmia caused by aDrosophila tropomyosin mutation is revealed using a novel optical coherencetomography instrument. PLoS One 5(12): e14348, 2010. doi:10.1371/journal.pone.0014348.

27. Nawaratne V, Leach K, Suratman N, Loiacono RE, Felder CC,Armbruster BN, Roth BL, Sexton PM, Christopoulos A. New insightsinto the function of M4 muscarinic acetylcholine receptors gained using anovel allosteric modulator and a DREADD (designer receptor exclusivelyactivated by a designer drug). Mol Pharmacol 74(4): 1119–1131, 2008.doi:10.1124/mol.108.049353.

28. Nebigil CG, Choi DS, Dierich A, Hickel P, Le Meur M, Messaddeq N,Launay JM, Maroteaux L. Serotonin 2B receptor is required for heartdevelopment. Proc Natl Acad Sci U S A 97(17): 9508–9513, 2000.doi:10.1073/pnas.97.17.9508.

29. Nichols CD, Roth BL. Engineered G-protein coupled receptors arepowerful tools to investigate biological processes and behaviors. FrontMol Neurosci 2: 16, 2009. doi:10.3389/neuro.02.016.2009.

30. Nichols CD. Drosophila melanogaster neurobiology, neuropharmacol-ogy, and how the fly can inform central nervous system drug dis-covery. Pharmacol Ther 112(3): 677–700, 2006. doi:org/10.1016/j.pharmthera.2006.05.012.

1664 Pharmacogenetic Inhibition of the Larval Heart • Majeed ZR et al.

J Appl Physiol • doi:10.1152/japplphysiol.00849.2013 • www.jappl.org

31. Nichols DE, Nichols CD. Serotonin receptors. Chem Rev 108(5): 1614–1641, 2008. doi:10.1021/cr078224o.

32. Obosi LA, Schuette DG, Europe-Finner GN, Beadle DJ, Hen R, KingLA, Bermudez I. Functional characterisation of the Drosophila 5-HTdro1and 5-HTdro2B serotonin receptors in insect cells: Activation of a G(al-pha) s-like protein by 5-HTdro1 but lack of coupling to inhibitoryG-proteins by 5-HTdro2B. FEBS Lett 381(3): 233–236, 1996.

33. Pandey UB, Nichols CD. Human disease models in Drosophila melano-gaster and the role of the fly in therapeutic drug discovery. Pharmacol Rev63(2): 411–436, 2011. doi:10.1124/pr.110.003293.

34. Ray RS, Corcoran AE, Brust RD, Kim JC, Richerson GB, Nattie E,Dymecki SM. Impaired respiratory and body temperature control uponacute serotonergic neuron inhibition. Science 333(6042): 637–642, 2011.doi:10.1126/science.1205295.

35. Samet MK, Rutledge CO. Antagonism of the positive chronotropic effectof norepinephrine by purine nucleosides in rat atria. J Pharmacol Exp Ther232(1): 106–110, 1985.

36. Saudou F, Boschert U, Amlaiky N, Plassat JL, Hen R. A family ofDrosophila serotonin receptors with distinct intracellular signaling prop-erties and expression patterns. EMBO J 11(1): 7–17, 1992.

37. Titlow JS, Rufer JM, King KE, Cooper RL. Pharmacological analysisof dopamine modulation in the Drosophila melanogaster larval heart.Physiol Rep 1(2): 1–9, 2013. doi:10.1002/phy2.20.

38. Urban JD, Clarke WP, von Zastrow M, Nichols DE, Kobilka B,Weinstein H, Javitch JA, Roth BL, Christopoulos A, Sexton PM,Miller KJ, Spedding M, Mailman RB. Functional selectivity and clas-sical concepts of quantitative pharmacology. J Pharmacol Exp Ther320(1): 1–13, 2007.

39. Vogler G, Bodmer R, Akasaka T. Drosophila model of congenital heartdiseases, congenital heart disease-selected aspects, P. Syamasundar Rao (Ed.).Available from: http://www.intechopen.com/books/congenital-heart-disease-selected-aspects/drosophila-model-of-congenital-heart-diseases, 2012. doi:10.5772/26739.

40. Witz P, Amlaiky N, Plassat JL, Maroteaux L, Borrelli E, Hen R.Cloning and characterization of a Drosophila serotonin receptor thatactivates adenylate cyclase. Proc Natl Acad Sci U S A 87(22): 8940–8944,1990.

41. Zornick E, Paisley K, Nichols R. Neural transmitters and a peptidemodulate Drosophila heart rate. Peptides 20(1): 45–51, 1999. http://dx.doi. org/10.1016/S0196-9781(98)00151-X.

1665Pharmacogenetic Inhibition of the Larval Heart • Majeed ZR et al.

J Appl Physiol • doi:10.1152/japplphysiol.00849.2013 • www.jappl.org